User:Chloe Paul/Sandbox 1

IntroductionIntroduction

Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its symmetric and asymmetric nature, its ability to bind DNA and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family) (Pfam); RTP is often compared to another protein with a similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). Its structural properties have proven to be integral to its function; as it must be able to bind DNA and have polarity (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction and yet be “permissive” at the other end.

The Structure of RTPThe Structure of RTP

RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination (Bussiere, 1995). These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form the dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and the contributing to the hydrophobic core of the other monomer (residue 122). Both monomers still remain structurally similar when they form the dimer in solution. It should be noted that when in solution the flexible loop between β2 and β3 are able to assume different conformations. (Bussiere, 1995)

RTP binding to DNARTP binding to DNA

RTP has been of interest in terms of its specific binding to DNA because it doesn’t use the common DNA structural motifs such as a basic leucine zipper, zinc finger or helix-turn-helix motif. It has been established that RTP like Tus, is sequence specific, as it binds as the Ter sites (comprising of two sequences that are imperfect inverted repeats (Vivian et al, 2007 and Bussiere 1995)). This means that RTP needs to be able to recognise specific bases in the helical DNA structure by reading the exposed edges of the bases located in the major and minor grooves of DNA not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA (Wilce et al, 2001).

The RTP:DNA interaction has been shown to be able to induce two different conformations of RTP depending upon the nature of the DNA. Early experiments used to determine how they interacted, used palindromic/symmetric DNA (sDNA) which was demonstrated to maintain the symmetry of RTP. However in nature, RTP was found to have a polar mechanism leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native/non-symmetric DNA (nDNA) that it induced an asymmetric form of RTP with a two faces. One face is described as being the “permissive” face as it allows the replication fork when it approaches this face first to proceed through. Whilst the other face, known as the “blocking” face demonstrates the action of terminating the approaching replication fork. It is the concept of these two faces that give rise to the polar mechanism of RTP.

The Fork Arrest MechanismThe Fork Arrest Mechanism

As previously noted the role of RTP is to terminate replication of the bacterial chromosome. It was originally assumed that the role of RTP was simply to arrest the replication fork allowing the DNA to cleanly separate (Wake 1997.) The proposed mechanism noted that the replication fork is only able to disrupt the RTP/ter interaction when approaching the A-site. This explains the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between the bound RTP and the replication fork helicase. The results of this research have confirmed a RTP/DnaB interaction in vivo, further suggesting this interaction plays an important role in replication fork arrest (Gautam 2001.) This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP which arrests the replication fork when it approaches from the appropriate direction (Kaplan 2009.)